New Benzothiazinone Derivatives and Their Use as Antibacterial Agents

ABSTRACT

The present invention relates to novel benzothiazinone derivatives and their use as antibacterial agents in infectious diseases of mammals (humans and animals) caused by bacteria, especially diseases like tuberculosis (TB) and leprosy caused by mycobacteria. The present invention aims at the generation of new compounds with activity against mycobacteria as potential new tuberculosis drugs to overcome problems concerning resistance and drug intolerance. The solution of the present invention is a compound of formula I 
     
       
         
         
             
             
         
       
     
     wherein R 1  to R 6  are as defined in the specification.

RELATEDNESS OF THE APPLICATION

The subject application is a continuation of co-pending U.S. Ser. No.12/227,655, filed Feb. 25, 2009, which is a 35 USC §371 ApplicationSerial No. PCT/EP2006/004942, filed May 24, 2006. Applicants herebyincorporate by reference in their entirety the foregoing applications,and all references cited or discussed herein.

BACKGROUND OF THE INVENTION

The present invention relates to novel benzothiazin derivatives andtheir use as antibacterial agents in infectious diseases of mammals(humans and animals) caused by bacteria, especially diseases liketuberculosis (TB) and leprosy caused by mycobacteria.

Thiazinone, there derivatives and their use as antibacterial agents,especially against mycobacteria (TB), laid open for public use in AR 2425 67 A1, AU 37 04 400 A1, CA 13 22 551 C1 or EP 0 245 901 B1 forinstance.

As known, there is a threadful worldwide increase in tuberculosisinfections with mycobacteria which developed resistance against theavailable therapeutics (B. R. Bloom, J. L Murray, Tuberculosis:commentary on a reemergent killer Science 257, 1992, 1055-1064).Extremely dangerous is the development of multidrug resistant (MDR)mycobacteria. These are mycobacteria, resistant at least against two ofthe most active tuberculosis drugs, isoniazid and rifampicin, but alsoagainst streptomycin, pyranzinamid and ethambutol. The proportion ofMDR-TB in some countries is already more than 20%. Together with theincreased number of TB diseases generally, worldwide it causes about3,000,000 deaths annually.

For the treatment of such diseases, like (TB) or leprosy there is anurgent need for new drugs with new mechanisms of actions, especially toovercome drug resistance and to overcome the known dramatic side effectsof the available drugs.

SUMMARY OF THE INVENTION

The present invention aims at the generation of new compounds withactivity against mycobacteria as potential new tuberculosis drugs toovercome problems concerning resistance and drug intolerance.

This aim has been solved by providing compounds of the formula I

Wherein R¹ and R² are, independently each from other, NO₂, CN, CONR⁷R⁸,COOR⁹, CHO, halogen, NR⁷R⁸, SO₂NR⁷R⁸, SR⁹, OCF₃, mono-, di ortrifluoromethyl;

R³ and R⁴ are, independently each from other, H, a saturated orunsaturated, linear or branched aliphatic radical having 1-7 chainmembers, cycloalkyl having 3-6 carbon atoms, benzyl, SR⁹, OR⁹;

R⁵ and R⁶ are, independently each from other, a saturated orunsaturated, halogenated or unhalogenated, linear or branched aliphaticradical having 1-8 chain members, cycloalkyl having 3-6 carbon atoms,phenyl, or R⁵ and R⁶ together represent a bivalent radical —(CR⁹₂)_(m)—, or R⁵ and R⁶ together represent bivalent radicals:

wherein m is 1-4, or represent bivalent radicals a saturated orunsaturated mono or polyheterocycles with heteroatoms N, S, O andsubstituted by (R¹⁰)x, wherein x is 1-4;

R⁷, R⁸ and R⁹ are, independently each from other H or a saturated orunsaturated, halogenated or unhalogenated, linear or branched aliphaticradical having 1-7 chain members, mono-, di or trifluoromethyl, halogen,phenyl, or R³ and R⁴ together represent a bivalent radical —(CH₂)_(n)—wherein n is 2-7;

R¹⁰ is H or a saturated or unsaturated, halogenated or unhalogenated,linear or branched aliphatic radical having 1-7 chain members, NO₂,NR⁷R⁸, CN, CONR⁷R⁸, COOR⁹, CHO, halogen, SO₂NR⁷R⁸, SR⁹, OR⁹, OCF₃,mono-, di or trifluoromethyl, benzyl or phenyl.

In a preferred embodiment the invention concerns compounds of theformula (I) selected from the group consisting of

-   2-(4-R⁵-4-R⁶-piperidin-1-yl)-8-nitro-6-trifluoromethyl-1,3-benzothiazin-4-one,-   6-cyano-2-(4-R⁵-4-R⁶-piperidin-1-yl)-8-nitro-1,3-benzothiazin-4-one,-   6-amido-2-(4-R⁵-4-R⁶-piperidin-1-yl)-8-nitro-1,3-benzothiazin-4-one,-   2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-R¹-6-R²-1,3-benzothiazin-4-one,-   2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-R¹-6-R²-1,3-benzothiazin-4-one,-   2-[(2R)-2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-R¹-6-R²-1,3-benzothiazin-4-one,-   2-[(2S)-2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-R¹-6-R²-1,3-benzothiazin-4-one,-   2-(2,3-dimethyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-R¹-6-R²-1,3-benzothiazin-4-one,-   2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-R¹-6-R²-1,3-benzothiazin-4-one,    wherein R¹, R², R⁵ and R⁶ have the above meanings,

The present invention is even more particularly concerned with at leastone compound selected from the group consisting of

-   2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one,-   2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one,-   2-(4,4-diethoxypiperidin-1-yl)-6,8-dinitro-1,3-benzothiazin-4-one,    7-methyl-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one,-   2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,-   2-(2,3-dimethyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,-   2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,-   2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile,-   2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile,-   8-amino-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-4-oxo-1,3-benzothiazine-6-carbonitrile    and-   8-amino-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6-(trifluoromethyl)-1,3-benzo-thiazin-4-one.

For the synthesis of the aimed compounds we developed our originalmethod of 1,3-benzothiazin-4-one synthesis with usage of dithiocarbamatederivatives as intermediate (method A). The classical method of1,3-benzothiazin-4-one synthesis with usage of thiocyanate salts (methodB) is usable too. Both are presented in the scheme below.

Surprisingly the compounds of the invention exhibit strong antibacterialactivity, especially against mycobacteria with minimal inhibitoryconcentrations (MIC) in the range of 0.23 μg/ml->10 μg/ml for fastgrowing mycobacteria, of 0.195-1.56 μg/ml for M. tuberculosis, includingmultiresistant strains determined by the classical method and of 0.030μg/ml for M. tuberculosis H37Rv determined by the Alamar Blue method.Surprisingly the compounds of the invention demonstrate a high level ofselectivity for mycobacteria only which reduces the potential foradverse side effects dramatically.

The compounds of the invention are non-mutagenic at 5 mg/ml in the SOSchromotest.

The compounds of the invention are in vivo therapeutically active in themurine model of tuberculosis infection superior compared to the mainantituberculosis drug isoniazid used as a positive control. 100% of micesurvived. All control animals died until day 33.

The compound of the invention (especially compound no 2=example 1 in theembodiments), is non toxic after per os administration of doses rangingup to 2000 mg/kg was the compound was well endured by animals in thefirst and 24 next coming hours after introducing. During 7 days ofinvestigations the compound 2 did not cause changes in general state andbehavior of the mice, it did not affect motor and reflex activity,active and calm cycles, grooming, food consumption, there were no casesof animal death. LD₅₀ for compound 2 is >2000 mg/kg.

Thus, the compounds of the invention are useful for the treatment oftubercular infection and other mycobacterial infections, in humans andin animals.

Accordingly, the invention concerns pharmaceutical compositionscomprising a compound of the formula I.

The invention relates furthermore to a compound of the formula I for usein a method for the treatment of bacterial infections in mammals.Preferred compounds of the formula I for use in such method are thosespecifically listed above.

The compounds of the invention are formulated for use by preparing adilute solution or suspension in pharmaceutically acceptable aqueous,organic or aqueous-organic medium for topical or parenteraladministration by intravenous, subcutaneous or intramuscular injection,or for intranasal application; or are prepared in tablet, capsule oraqueous suspension form with conventional excipients for oraladministration or as suppositorium.

The compounds can be used in dosages from 0.001-1000 mg/kg body weight.

The examples which follow in the subsequent experimental part serve toillustrate the invention but should not be construed as a limitationthereof.

The structures of the compounds of the invention were established bymodes of synthesis and elementary analysis, and by nuclear magneticresonance and/or mass spectra, as well as by X-ray analysis.

DETAILED DESCRIPTION OF THE INVENTION Starting Materials

Chemicals and solvents were purchased from Lancaster Synthesis(Lancashire, England) or from Aldrich (Sigma-Aldrich Company, St-Louis,US) and were used in the synthesis without additional purification.Melting points were determined according to the BP procedure and areuncorrected (Electrothermal 9001, GB). If analyses are indicated only bythe symbols of the elements, analytical results are within ±0.3% of thetheoretical values (Carlo-Erba 5500, Italy). NMR spectra were determinedwith a Varian Unity Plus 400 (USA). Shifts for ¹H NMR are reported inppm downfield from TMS (δ). Mass spectra were obtained using a FinniganSSQ-700 (USA) instrument with direct inject. Reactions and purity ofcompounds were controlled by TLC with usage Silicagel 60 F₂₅₄ aluminiumsheets (Merck Co, Germany).

Example 12-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,(compound 1) Method A.

To a stirred 50 mL solution of 25% aqueous ammonia was added drop-wise asolution of 5 g of 2-chloro-3-nitro-5-trifluoromethylbenzoyl chloride(D. E Welch, R. R. Baron, B. A. Burton, J. Med. Chem. 12; 2; 1969;299-303) in acetonitrile (10 mL) at −20° C. 10 min later, 50 ml of ethylacetate was added. The organic phase was separated, washed twice inwater, dried over Na₂SO₄, treated by activated carbon, filtered andconcentrated in vacuum. The crude product was purified bycrystallization from ethanol. The yield of2-chloro-3-nitro-5-(trifluoromethyl)benzamide was 92%. mp 195-197° C.(methanol).

Anal. Calcd. for C₈H₄ClF₃N₂O₃: C, 35.78; H, 1.50; N, 10.43.

Found: C, 36.01; H, 1.53; N, 10.39.

0.5 g of 2,2-chloro-3-nitro-5-(trifluoromethyl)benzamide was dissolvedin a 25 ml of ethanol. The reaction mixture was treated with of 0.5 g of1,4-dioxa-8-azaspiro[4.5]decane-8-carbodithioic acid sodium saltdihydrate (Z. Ge, R. Li, T. Cheng, Synth. Commun., 29, 18, 1999,3191-3196) and stored for 18 h at room temperature. It was then pouredinto 50 ml of cooled water and the resulting yellow precipitate wasfiltered off. Pure final product was obtained after recrystallizationtwice from ethanol.2-(Aminocarbonyl)-6-nitro-4-(trifluoromethyl)phenyl-1,4-dioxa-8-azaspiro[4.5]decane-8-carbodithioateis light yellow crystalline solid. Yield 0.47 g %. mp 138-140° C.

Anal. Calcd. for C₁₁H₁₂N₄O₂S₂: C, 42.57; H, 3.57; N, 9.31; S, 14.21.

Found: C, 42.61; H, 3.67; N, 9.22; S, 14.30.

0.4 g of2-(aminocarbonyl)-6-nitro-4-(trifluoromethyl)phenyl-1,4-dioxa-8-azaspiro[4.5]decane-8-carbodithioatewas dissolved in a 25 ml of ethanol. The reaction mixture was treatedwith of 0.32 g of Na₂HPO₄×12H₂O and refluxed for 6 h. It was then cooledand light yellow precipitate was filtered off and washed by 30 mlmethanol. Pure final product was obtained after recrystallization twicefrom ethanol.2-(1,4-Dioxa-8-aza-spiro[4.5]dec-8-yl)-8-nitro-6-trifluoromethyl)-1,3-benzothiazin-4-oneis light yellow crystalline solid. Yield 0.47 g %. mp 211-212° C.

R_(f) ((hexane-acetone; 2/1)-0.35

MS m/z 417 (M⁺).

¹H NMR (DMSO-d₆) δ 8.83 and 8.77 (two 1H, two s, 2CH), 3.80 (8H, broads, N(CH₂CH₂)₂C), 2.02 (4H, broad s, OCH₂CH₂O) ppm.

Anal. Calcd. for C₁₆H₁₄F₃N₃O₅S: C, 46.04; H, 3.38; N, 10.07; S, 7.68.

Found: C, 45.94; H, 3.37; N, 10.09; S, 7.76.

Method B. The procedure in detail was the same as described in J.Imrich, P. Kristian, Coll. Czech. Chem. Commun., 47, 1982, 3268-3282; D.Koscik, P. Kristian, J. Gonda, E. Dandarova, Coll. Czech. Chem. Commun.,48, 1983, 3315-3328; D. Koscik, P. Kristian, O. Forgac, Coll. Czech.Chem. Commun., 48, 1983, 3427-3432; T. H. Cronin, H.-J. E. Hess, U.S.Pat. No. 3,522,247. Yield of2-(1,4-dioxa-8-aza-spiro[4.5]dec-8-yl)-8-nitro-6-trifluoromethyl)-1,3-benzo-thiazin-4-oneis 0.21%. The compound is identical by spectroscopical data to thecompound synthesized by method A.

Example 22-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoro-methyl)-1,3-benzothiazin-4-one,(compound 2)

Following the procedure of Example 1. Light yellow crystalline solid.

Yield 54%. mp 192-3° C.

R_(f) (hexane-acetone; 2/1)-0.30.

MS m/z 431 (M^(t)).

¹NMR (DMSO-d₆) δ 8.81 and 8.77 (two 1H, two s, 2CH), 4.24 (1H, m, CH),4.11 (1H, m, CH), 4.06 (4H, broad s, N(CH₂)₂), 3.47 (1H, t, CH), 3.27(1H, s, CH), 1.80 (4H, broad d, C(CH₂)₂), 1.23 (3H, d, CH₃) ppm.

Anal. Calcd. for C₁₇H₁₆N₃O₅S: C, 47.33; H, 3.74; N, 9.74; S, 7.43.

Found: C, 47.36; H, 3.80; N, 9.87; S, 7.51.

Example 32-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one,(compound 4)

Following the procedure of Example 1 with usage of2-hydroxy-3,5-dinitrobenzoic acid as starting material. Light yellowcrystalline solid.

Yield 43%. mp 271-3° C. (EtOH/DMF).

R_(f) (hexane-acetone; 2/1)-0.25.

MS m/z 394 (M⁺).

¹H NMR (DMSO-d₆) δ 9.15 and 9.12 (two 1H, two s, 2CH), 3.86 (8H, broads, N(CH₂CH₂)₂C), 2.97 (4H, broad s, OCH₂CH₂O) ppm.

Anal. Calcd. for C₁₅H₁₄N₄O₇S: C, 45.68; H, 3.58; N, 14.21; S, 8.13.

Found: C, 45.34; H, 3.56; N, 14.30; S, 7.98.

Example 42-(2-methyl-1,4-dioxa-8-azaspiro[4.5]decyl)-6,8-dinitro-1,3-benzothiazin-4-one,(compound 4)

Following the procedure of Example 1 with usage of2-hydroxy-3,5-dinitrobenzoic acid as starting material. Yellowcrystalline solid. Yield 57%. mp 139-142° C. (EtOH/DMF).

R_(f)(hexane-acetone; 2/1)-0.50.

MS m/z 408 (M⁺).

¹H NMR (DMSO-d₆) δ 9.08 and 9.11 (two 1H, two s, 2CH), 4.23 (1H, m, CH),4.10 (1H, m, CH), 4.06 (4H, broad s, N(CH₂)₂), 3.43 (1H, t, CH), 3.27(1H, s, CH), 1.80 (4H, broad d, C(CH₂)₂), 1.20 (3H, d, CH₃) ppm.

Anal. Calcd. for C₁₆H₁₆N₄O₇S: C, 47.06; H, 3.95; N, 13.72; S, 7.85.

Found: C, 46.87; H, 3.91; N, 13.57; S, 7.83.

Example 52-(2,3-dimethyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,(compound 5)

Following the procedure of Example 1 with usage of2-hydroxy-3-nitro-5-trifluoromethylbenzoic acid as starting material.Light yellow crystalline solid. Yield 58%. mp 205-207° C. (EtOH/DMF).

R_(f) (hexane-acetone; 2/1)-0.55.

MS m/z 44522 (M⁺).

¹NMR (DMSO-d₆) δ 8.82 and 8.77 (two 1H, two s, 2CH), 3.86 (4H, broad c,N(CH₂)₂), 3.45-3.53 (2H, m, 2CH), 2.41 (4H, broad d, C(CH₂)₂), 1.13-1.17(6H, m, 2CH₃) ppm.

Anal. Calcd. for C₁₈H₁₈F₃N₃O₅S: C, 48.54; H, 4.07; N, 9.43; S, 7.20.

Found: C, 48.66; H, 4.12; N, 9.32; S, 7.46.

Example 62-(4,4-diethoxypiperidin-1-yl)-6,8-dinitro-1,3-benzothiazin-4-one,(compound 6)

Following the procedure of Example 1 with usage as starting material2-hydroxy-3,5-dinitrobenzoic acid. Yellow crystalline solid. Yield 32%.mp 179-181° C. (i-PrOH).

R_(f)(hexane-acetone; 2/1)-0.30.

MS m/z 424 (M⁺).

¹H NMR (DMSO-d₆) δ 9.08 and 9.11 (two 1H, two s, 2CH), 3.60-3.67 (4H, m,N(CH₂)₂) 2.11-2.08 (4H, m, C(CH₂)₂), 3.47 and 3.57 (two 2H, q, 2OCH₂),1.16 (6H, t, 2CH₃), ppm.

Anal. Calcd. for C₁₇H₂₀N₄O₇S: C, 48.11; H, 4.75; N, 13.20; S, 7.56.

Found: C, 48.12; H, 4.73; N, 13.41; S, 7.67.

Example 72-(7,12-dioxa-3-azaspiro[5.6]dodec-3-yl)-6,8-dinitro-1,3-benzothiazin-4-one,(compound 7)

Following the procedure of Example 1 with usage as starting material2-hydroxy-3,5-dinitrobenzoic acid. Yellow crystalline solid. Yield 51%.mp 193-195° C. (i-PrOH/DMF).

R_(f) (hexane-acetone; 2/1)-0.45.

MS m/z 422 (M⁺).

¹H NMR (DMSO-d₆) δ 8.97 and 9.16 (two 1H, two s, 2CH), 3.57-3.74 (8H, m,4-CH₂), 1.93-2.35 (8H, m, 4-CH₂) ppm.

Anal. Calcd. for C₁₇H₁₈N₄O₇S: C, 48.34; H, 4.30; N, 13.26; S, 7.56.

Found: C, 48.21; H, 4.43; N, 13.30; S, 7.66.

Example 82-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-7-methyl-6,8-dinitro-1,3-benzothiazin-4-one,(compound 8)

Following the procedure of Example 1 with usage as starting material2-hydroxy-4-methyl-3,5-dinitrobenzoic acid. Yellow crystalline solid.

Yield 51%. mp 207-210° C. (i-PrOH/DMF).

R_(f) (hexane-acetone; 2/1)-0.30.

MS m/z 408 (M⁺).

¹H NMR (DMSO-d₆) δ 8.77 (1H, s, CH), 3.86 (8H, broad s, N(CH₂CH₂)₂C),2.97 (4H, broad c, OCH₂CH₂O), 2.79 (3H, s, CH₃) ppm.

ppm.

Anal. Calcd. for C₁₆H₁₆N₄O₇S: C, 47.06; H, 3.95; N, 13.72; S, 7.85.

Found: C, 47.12; H, 4.01; N, 13.69; S, 7.94.

Example 92-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile,(compound 9)

To a stirred solution of 5 g (19 mmol) 2-hydroxy-5-iodobenzoic acid in50 ml DMF was added by small portions dry 2.5 g (22 mmol) of CuCN (I).The reaction mixture was refluxed during 5 h, 100 ml of water and 50 mlethylacetate were added. After it conc. Hydrochloric acid was added upto pH˜3 very carefully under good ventilation. The organic phase wasseparated, washed twice in water, dried over Na₂SO₄, treated byactivated carbon, filtered and concentrated in vacuum. The crude productwas purified by crystallization from water. The yield of5-cyano-2-hydroxybenzoic acid was 71%. Following the procedure ofExample 1. Yield 44%. mp 217-220° C. (EtOH/DMF).

R_(f) (hexane-acetone; 2/1)-0.50.

MS m/z 374 (M⁺).

¹H NMR (DMSO-d₆) δ 8.74 and 8.67 (two 1H, two s, 2CH), 3.41 (8H, broads, N(CH₂CH₂)₂C), 2.93 (4H, broad s, OCH₂CH₂O) ppm.

Anal. Calcd. for C₁₆H₁₄N₄O₅S: C, 51.33; H, 3.77; N, 14.97; S, 8.57.

Found: C, 51.30; H, 3.84; N, 14.89; S, 8.62.

Example 102-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile,(compound 10)

Following the procedure of Example 9. Yellow crystalline solid. Yield34%. mp 251-253° C. (EtOH/DMF).

R_(f) (hexane-acetone; 2/1)-0.40.

MS m/z 388 (M⁺).

¹H NMR (DMSO-d₆) δ 8.73 and 8.61 (two 1H, two s, 2CH), 4.23 (1H, m, CH),4.11 (1H, m, CH), 4.07 (4H, broad s, N(CH₂)₂), 3.51 (1H, t, CH), 3.27(1H, s, CH), 1.81 (4H, broad d, C(CH₂)₂), 1.22 (3H, d, CH₃) ppm ppm.

Anal. Calcd. for C₁₇H₁₆N₄O₅S: C, 52.57; H, 4.15; N, 14.43; S, 8.26.

Found: C, 52.42; H, 4.08; N, 14.50; S, 8.27.

Example 112-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile,(compound 11)

Following the procedure of Example 9. Yellow crystalline solid. Yield40%. mp 230-232° C. (EtOH/DMF).

R_(f) (hexane-acetone; 2/1)-0.15.

MS m/z 388 (M⁺).

¹H NMR (DMSO-d₆) δ 8.74 and 8.61 (two 1H, two s, 2CH), 3.29-3.65 (6H, m,3CH₂), 2.38 (4H, broad s, 2CH₂), 1.82-1.93 (4H, m, 2CH₂) ppm.

Anal. Calcd. for C₁₇H₁₆N₄O₅S: C, 52.57; H, 4.15; N, 14.43; S, 8.26.

Found: C, 52.52; H, 4.11; N, 14.59; S, 8.13.

Example 12

Determination of the in vitro inhibitory activity of the compounds ofthe invention against mycobacteria.

The antibacterial activities of the compounds against Mycobacteriumsmegmatis SG 987, M. aureum SB66, M. vaccae IMET 1010670 and M.fortuitum B were tested by determination of minimal inhibitoryconcentrations (MIC) by the micro broth dilution method inMueller-Hinton broth (Difco) according to the NCCLS guidelines [NationalCommittee for Clinical Laboratory Standards: Methods for dilutionantimicrobial susceptibility tests for bacteria that grow aerobically;5^(th) Ed.; Villanova, Ed.; Approved standard Document M7-A5. NCCLS,(2000)].

Activity against M. tuberculosis H37Rv was tested by the followingmethod for determination of minimal inhibitory concentrations (MIC) andminimal bactericidal concentrations (MBC):

Strains were inoculated onto solid Lowenstein-Jensen medium. After 21days, the cultures grown were used to prepare an inoculum suspensioncorresponding to 5×10⁸ microbial cells/ml). With 0.2 ml of thatsuspension tubes with 2 ml liquid Shkolnikova medium, containingcorresponding concentrations of compounds under study—from 100.0 to0.195 mg/ml, were inoculated. After 14 days of incubation at 37° C. thetubes with liquid medium were centrifuged for 15 min. at 3000 RPM.

After discarding the supernatant, the sediment was resuspended in 0.8 mlof sterile 0.9% NaCl. 0.1 ml of the suspension was used to preparesmears subsequently stained by the Ziehl-Neelsen method. The remainingsediment was inoculated in 0.2 ml volumes into three tubes with soliddrug free Lowenstein-Jensen medium to determine minimal bactericidalconcentrations (MBC). The results were read after 21-28 days ofcultivation at 37° C. Controls were tubes cultured with test-strains nottreated with the studied agents.

Minimal bactericidal concentration of drugs (MBC) was considered as thedrug concentration completely inhibiting the growth of mycobacteria onthe solid medium. The bacteriostatic effect (MIC) was characterized bythe presence of only individual mycobacteria in the smear and a strongdecrease in the number of colonies grown on solid media compared to thecontrols.

The results are presented in Tables 1 and 2.

TABLE 1 Antimicrobial activity of compounds as of the formula Idetermined by minimal inhibitory concentrations MIC [μg/ml] Compound. M.smegmatis M. vaccae M. fortuitum 1 12.5 ng/ml 3.12 ng/ml 12.5 ng/ml 21.56 ng/ml 0.76 pg/ml 0.023 pg/ml 3 0.2 μg/ml 0.0015 μg/ml 0.006 μg/ml 40.2 μg/ml 0.003 μg/ml 0.003 μg/ml 5 6.25 ng/ml 0.078 ng/ml 0.078 ng/ml6 >10 μg/ml 0.04 μg/ml 0.08 μg/ml 7 0.78 μg/ml 0.003 μg/ml 0.003 μg/ml 80.4 μg/ml 0.025 μg/ml 0.025 μg/ml 9 0.05 μg/ml 3.12 ng/ml 25 ng/ml 10 25ng/ml 3.12 ng/ml 12.5 ng/ml 11 0.05 μg/ml 6.25 ng/ml 25 ng/ml

TABLE 2 Antimicrobial activity of compounds of the formula I againstMycobacterium tuberculosis H37Rv and clinical isolates 6341 and 6374 asdetermined by minimal inhibitory concentrations (MIC) and minimalbactericidal concentrations (MBC) MBC MIC MBC MIC (μg/mL) (μg/mL) StrainCompound (μg/mL) (μg/mL) mean mean H37Rv 10 0.58 0.39 0.71 0.45 63410.78 0.58 6374 0.78 0.39 H37Rv 9 0.29 0.195 0.75 0.52 6341 1.17 0.786374 0.78 0.58 H37Rv 2 0.58 0.39 0.45 0.29 6341 0.39 0.29 6374 0.390.195 H37Rv 5 0.58 0.39 0.45 0.39 6341 0.39 0.39 6374 0.39 <0.39 H37Rv 10.58 0.39 1.75 1.17 6341 2.34 1.56 6374 2.34 1.56 H37Rv Isoniazid 1.150.97 1.15 0.97 6341 (INH) >100 >100 Not active, >100 6374 >100 >100 Notactive, >100

Example 13

Determination of the in vivo inhibitory activity of the compounds of theinvention against Mycobacterium tuberculosis in the murine TB model

To determine the chemotherapeutic efficacy we used BALB/c line mice withexperimental hematogenously disseminated tuberculosis. The mice wereobtained from the Central Animal Nursery of the Russian Academy ofMedical Sciences. In this study we included mice after quarantine,standardized by weight (20-25 g) and male only. The mice were infectedwith a 2-week virulent culture of Mycobacterium tuberculosis H37Rv byintravenous injection (into tail vein) of the mycobacterial suspensionat a dose of 5×10⁶ CFU (Colony Forming Unit) in 0.5 ml saline. All theexperimental animals were divided into groups depending on the treatmentregimen used (Table 3). Tested drug doses were selected based on thedata from literature and on results of previous investigations.

TABLE 3 No Dose Number of animals group Compound (mg/kg) per group 3 212 10 4 2 25 10 5 Isoniazid (INH) 25 10 6 without treatment 10

Treatment was started the next day after infection. The drugs wereintroduced orally as suspension in carboxymethylcellulose/water with asmall quantity PEG-400.

Chemotherapy was administered daily 6 times per week (except Sunday).

The animals were killed with ether narcosis. To determine the efficacyof each treatment regimen we registered macroscopical changes inparenchymal organs of the mice, growth of mycobacteria from pathologicmaterial on solid media, as well as a bacterioscopical index of organinjury. We carried out a qualitative and quantitative analysis ofmacroscopical changes in the liver, spleen and lungs and calculated aninjury index (using a four-score scale).

Macroscopical evaluation of the efficacy of each treatment regimen wasexpressed in the efficacy index, calculated using a formula.

${{Efficacy}\mspace{14mu} {idex}} = {{100\%} - {\frac{\begin{matrix}{{Injury}\mspace{14mu} {index}\mspace{14mu} {of}\mspace{14mu} {the}} \\{{studied}\mspace{14mu} {group}}\end{matrix}}{\begin{matrix}{{Injury}\mspace{14mu} {index}\mspace{14mu} {of}\mspace{14mu} {the}} \\{{control}\mspace{14mu} {group}}\end{matrix}} \times 100}}$

Microbiological examination included culture for determination of CFU inparenchymal organs. For this purpose, we homogenisated the right lungand separately the spleen with 6% sulfuric acid, centrifuged, washed bywater and saline. The yield (about 0.5 mL) was diluted by 1.0 mL ofsaline and homogenisated. This suspension (0.5 mL) of test organs wasdiluted 100 and 1000 times by saline and was distributed on solid Finn-2medium. The cultures were incubated at 37° C. for 1 months and readweekly starting from day 10. After 28 days CFU's were counted.

Data of macroscopical and microbiological examinations of parenchymalorgans of mice which died during the experiment were also considered inthe overall assessment of the experimental results which are representedin tables 4-6.

TABLE 4 Indexes of organ injury in mice and treatment efficacy DoseEfficacy index Group Drug (mg/kg) Injury index (%) 3 Compound 2 12 2.144.7 4 Compound 2 25 1.0 78 5 INH, Isoniazid 25 1.2 70.5 6 Control — 3.8—

TABLE 5 Results of microbiological examination of right lung and spleenof experimental mice (42 days after inoculation of the culture medium)right lung spleen Dose Culture, without Culture, without Group Compound(mg/kg) dilution CFU dilution CFU 3 2 12 ~60 ~60 4 2 25 ~35 ~35 5 INH,Isoniazid 25 ~40 ~40 6 Control — >120 >120 (total growth) (total growth)

TABLE 6 Survival of animals Day of Group 3 Group 4 Group 5 Group 6Treatment Compound 2 Compound 2 INH Control 1 10 10 10 10 2 10 10 10 103 10 10 10 10 4 10 10 10 10 5 10 10 10 10 6 10 10 10 10 7 10 10 10 10 810 10 10 10 9 10 10 10 10 10 10 10 10 10 11 10 10 10 10 12 10 10 10 1013 10 10 10  9 14 10 10 10  9 15 10 10 10  9 16 10 10 10  9 17 10 10 10 9 18 10 10 10  9 19 10 10 10  9 20 10 10 10  8 21 10 10 10  8 22 10 1010  8 23 10 10 10  8 24 10 10 10  8 25 10 10 10  5 26 10 10 10  4 27  100%   100%   100%    40% All control animals died until day 33

1. A compound of formula (1):

or a salt thereof, wherein R¹ and R² are, independently of each other,NO₂, CN, CONR⁷R⁸, CHO, halogen, NR⁷R⁸, SO₂NR⁷R⁸, SR⁹, OCF₃, mono-, di-or trifluoromethyl; R³ and R⁴ are, independently of each other, H, asaturated or unsaturated, linear or branched aliphatic radical having1-7 chain members, cycloalkyl having 3-6 carbon atoms, benzyl, SR⁹, OR⁹;R⁵ and R⁶ are, independently of each other, a saturated or unsaturated,linear or branched aliphatic radical having 1-8 chain members,cycloalkyl having 3-6 carbon atoms, phenyl, or R⁵ and R⁶ togetherrepresent a bivalent radical —(CR⁹ ₂)m-, or R⁵ and R⁶ together representa bivalent radical:

wherein m is 1-4, or represent bivalent radicals, a saturated orunsaturated mono or polyheterocyles with heteroatoms N, S, O, andsubstituted by)(R¹⁰)_(x), where x is 1-4; R⁷, R⁸ and R⁹ are,independently of each other, H or a saturated or unsaturated,halogenated or unhalogenated, linear or branched aliphatic radicalhaving 1-7 chain members, mono-, di- or trifluoromethyl, halogen,phenyl; R¹⁰ is H or a saturated or unsaturated, halogenated orunhalogenated, linear or branched aliphatic radical having 1-7 chainmembers, NO₂, NR⁷R⁸, CN, CONR⁷R⁸, COOR⁹, CHO, halogen, SO₂NR⁷R⁸, SR⁹,OR⁹, OCF₃, mono-, di- or trifluoromethyl, benzyl or phenyl.
 2. A2-(2,3-R⁵R⁶-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoro-methyl)-1,3-benzothiazin-4-oneof the formula (1) according to claim 1, wherein R¹ represents NO₂, R²is CF₃, R³ and R⁴ are H, and R⁵ and R⁶ have the meanings given inclaim
 1. 3. A2-(2,3-R⁵R⁶-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrileof the formula (1) according to claim 1, wherein R¹ represents NO₂, R²is CN, R³ and R⁴ are H, and R⁵ and R⁶ have the meanings given inclaim
 1. 4. A2-(2,3-R⁵R⁶-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-4-oxo-1,3-benzothiazineof the formula (1) according to claim 1, wherein R¹ and R² representNO₂, R³ and R⁴ are H, and R⁵ and R⁶ have the meanings given in claim 1.5. A2-(2,3-R⁵R⁶-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbamideof the formula (1) according to claim 1, wherein R¹ represents NO₂, R²is CONH₂, R³ and R⁴ are H, and R⁵ and R⁶ have the meanings given inclaim
 1. 6. A compound of the formula (1) according to claim 1 selectedfrom the group consisting of:2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazine-4-one,2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one,2-(4,4-diethoxypiperidin-1-yl)-6,8-dinitro-1,3-benzothiazin-4-one,7-methyl-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one,2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,2-(2,3-dimethyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile,2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile,8-amino-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-4-oxo-1,3-benzothiazine-6-carbonitrile,and8-amino-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6-(trifluoromethyl)-1,3-benzothiazin-4-one.7. The compound of the formula (1) according to claim 1, wherein R⁵ andR⁶ are alkyl.
 8. A pharmaceutical composition comprising a compound ofthe formula (1) according to claim
 1. 9. A method of therapeutically orprophylactically treating tuberculosis in a mammal, comprisingadministering to the mammal a therapeutic or prophyltic dose of apharmaceutical composition comprising a compound of formula (1)

Wherein R¹ and R² are, independently from each other, NO₂, CN, CONR⁷R⁸,CHO, halogen, NR⁷R⁸, SO₂NR⁷R⁸, SR⁹, OCF₃, mono-, di- or trifluoromethyl;R³ and R⁴ are, independently of each other, H, a saturated orunsaturated, linear or branched aliphatic radical having 1-7 chainmembers, cycloalkyl having 3-6 carbon atoms, benzyl, SR⁹, OR⁹; R⁵ and R⁶are, independently of each other, a saturated or unsaturated, linear orbranched aliphatic radical having 1-8 chain members, cycloalkyl having3-6 carbon atoms, phenyl, or R⁵ and R⁶ together represent a bivalentradical —(CR⁹ ₂)_(m)—, or R⁵ and R⁶ together represent a bivalentradical:

wherein m is 1-4, or represent bivalent radicals, a saturated orunsaturated mono or polyheterocyles with heteroatoms N, S, O, andsubstituted by (R¹⁰)_(X), where x is 1-4; R⁷, R⁸ and R⁹ are,independently of each other, H or a saturated or unsaturated,halogenated or unhalogenated, linear or branched aliphatic radicalhaving 1-7 chain members, mono-, di- or trifluoromethyl, halogen, orphenyl, or R³ and R⁴ together represent a bivalent radical —(CH₂)_(n)—wherein n is 2-7; R¹⁰ is H or a saturated or unsaturated, halogenated orunhalogenated, linear or branched aliphatic radical having 1-7 chainmembers, NO₂, NR⁷R⁸, CN, CONR⁷R⁸, COOR⁹, CHO, halogen, SO₂NR⁷R⁸, SR⁹,OR⁹, OCF₃, mono-, di- or trifluoromethyl, benzyl or phenyl.